DNA replication is a crucial step in the cell cycle where cellular DNA must be faithfully copied only once. Failure to do so will result in either incomplete replication or multiple rounds of replication, both of which result in aberrant chromosome numbers. This proposal aims to understand how a protein known as Cdt1, which has been shown in all organisms to be the key player in controlling DNA replication, is regulated. Mis-regulation of Cdt1 levels plays a key role in tumorigenesis, underscoring the importance of this pathway in cancer biology. PUBLIC HEALTH RELEVANCE: The ubiquitin proteasome system (UPS) plays a key role in cellular homeostasis by regulating both bulk protein turnover as well as the degradation of key regulatory proteins. Its role is of particular importance in regulating the spatial and temporal aspects of the cell cycle. Key cell cycle proteins are degraded at precise points in the cell cycle to signal that the next phase may be initiated. One such UPS target is the evolutionarily conserved DNA replication licensing factor Cdt1. Cdt1 licences origins of replication early in th G1 phase of the cell cycle. To ensure that DNA is replicated only once, Cdt1 must promptly be degraded via the UPS. Moreover, Cdt1 is also a target of the DNA damage response and is rapidly degraded when cells are treated with genotoxic agents to prevent replication initiation in the face of unrepaired DNA lesions. Surprisingly, canonical DNA damage kinases such as ATM/ATR that take part in many aspects of the cellular response to DNA damage, do not appear to be involved in its degradation. While Cul4-Ddb1Cdt2 has been identified to ubiquitinate Cdt1 that is associated with chromatin-bound PCNA, there is still not a complete catalog of genes in this pathway. In order to identify the entire repertoire of genes that impinge on Cdt1 turnover, I have developed and completed a high-throughput, microscopy-based, quantitative genome-wide loss-of-function screen in DNA damaged cells. This screen has identified not only all the known complexes that regulate Cdt1 turnover, but also a large number of novel genes that appear to stabilize Cdt1 in DNA damaged cells. In many cases, I have recovered multiple subunits of macro-molecular complexes, increasing the confidence that these complexes have bona fide roles in the the Cdt1 pathway. This proposal aims to dissect the mechanism by which one of the strongest complexes identified in the screen impact Cdt1 turnover. I will focus in on the mechanism by which the p97/Ufd1-Npl4-Ufd2 complex delivers ubiquitinated Cdt1 to the 26S proteasome. I identified p97 as well as its cofactors Ufd1 and Ufd2 from the screen. This complex has been shown to extract proteins from multimeric complexes and deliver the ubiquitinated target to the proteasome. I will identify all the components of this complex that are required for Cdt1 turnover and test if this complex associates with ubiquitinated Cdt1 on chromatin. Importantly, I will determine if p97 and cofactors extract Cdt1 from PCNA and chromatin via p97's AAA-ATPase activity using techniques in mammlian cells and Xenopus egg extracts. Finally, I will attempt to reconstitute the ubiquitination and degradation of Cdt1 biochemically with purified components to rigorously test all the predictions made by the model. In summary, this proposal aims to ascribe molecular function to new genes in the Cdt1 destruction pathway, mis-regulation of which has been demonstrated to result in tumorigenesis in multiple settings.